Regulation of conventional versus innate CD8+ T cell development in the thymus is now known to produce a wide array of distinct T cell lineages. Many of these T cell subsets play key roles in regulating immune responses, both to self as well as to pathogens. In addition to the conventional CD4+ and CD8+ ???T cells that are key components of the adaptive immune response, there are regulatory T cells, NKT cells, ?? T cells, and a variety of additional innate T cell subsets. The appropriate balance of T cells developing into each of these lineages is essential to maintain immunological homeostasis, self-tolerance, and the ability to produce both rapid and delayed responses to pathogenic infections. Currently, the molecular mechanisms governing these developmental lineage choices are under intense investigation. Our own studies have identified a signaling pathway involving the Tec family tyrosine kinase, Itk, which determines conventional versus innate CD8+ T cell development. In wild-type (WT) thymocytes with normal Itk function, MHC class I-specific T cells predominantly develop into conventional naive CD8+ T cells, which are precursors of effector cytotoxic T cells. In addition, a very minor subset of cells develop into innate CD8+ T cells that have characteristics of previously-activated memory CD8+ T cells, and exhibit immediate effector function when activated. In contrast to this, MHC class I-specific thymocytes lacking the Tec kinase, Itk, develop nearly exclusively into innate CD8+ T cells that express high levels of the T-box transcription factor, Eomesodermin. These findings indicate that a signaling pathway requiring Itk regulates the lineage decision between conventional and innate CD8+ T cells. As Itk is well known as a component of the TCR signaling pathway leading to phospholipase?-31 activation and actin polymerization, these data also implicate altered TCR signaling as a modulator of these key T cell lineage decisions. To determine the transcriptional regulators of this lineage decision, we performed a microarray experiment to identify factors differentially expressed between WT conventional and Itk-deficient innate CD8+ thymocytes. Interestingly, this analysis indicated that the single most highly up-regulated transcription factor in WT relative to Itk-deficient CD8+ thymocytes is IRF4; further, our preliminary studies indicate that in the absence of IRF4, nearly all CD8+ T cells also develop into the innate lineage. In contrast, the transcription factor most highly expressed in Itk- deficient thymocytes relative to WT is Runx2. We hypothesize that Itk signaling promotes conventional CD8+ T cell development by inducing the transcription of IRF4, and that in the absence of Itk, Runx2 upregulation converts conventional CD8+ T cells into innate T cells, leading to upregulation of Eomesodermin. To determine the importance of these transcription factors in regulating conventional versus innate CD8+ T cell development we propose to examine whether IRF4 is essential for conventional CD8+ T cell lineage commitment. We will also investigate whether IRF4 is sufficient to suppress Eomesodermin expression in CD8+ T cells. Third, we will determine whether different strengths of TCR signaling lead to graded expression of IRF4. Finally, we will examine whether Runx2 is required for innate CD8+ T cell development. PUBLIC HEALTH RELEVANCE: Our immune system protects us against a wide array of pathogens using many different types of white blood cells. The work described in this proposal will investigate how our body produces the correct types of white blood cells in the correct proportions. This understanding will aid in efforts to regulate and control the immune system to fight infections and eradicate cancer cells.